The broad objective of this project is to develop instrumentation and chemistry imaging technologies to advance the study of protein glycosylation in living subjects. Protein glycosylation is the most abundant and complex posttranslational modification (PTM). Changes in protein glycosylation have been correlated with cancer progression, neurological disorders and many other diseases. Moreover, glycans are dynamic in time, space and environment. Hence, in order to truly study the function of glycans in health and disease, their dynamic spatiotemporal behavior should be imaged in living subjects, and where relevant, in the context of the proteins they modify. However, current in vivo imaging tools have limited spatial and temporal resolutions and are not capable of visualizing protein glycosylations. We propose a novel molecular imaging modality that would allow imaging the spatiotemporal behavior of glycans in living murine cancer models. Such tools would advance the field of glycobiology, accelerate the discovery and validation of new disease biomarkers, and bridge traditional biochemistry with high-level biological disease models. Beyond the basic cancer biology prospects, imaging the tumor glycome may provide an indication on the tumor aggressiveness. Such information will guide treatment decisions of diseases such as prostate cancer (advise patients on prostatectomy versus active surveillance) as well as other cancer types. We will optimize a new imaging instrument we developed based on optical coherence tomography, to allow visualizing nanoparticle-based contrast agents in living tissues with ultrahigh sensitivities (Aim 1). Second, we will synthesize two new classes of nanoparticle imaging agents: the first for targeting and visualizing a specific glycan epitope (Aim 2a) and the second for targeting and visualizing a specific glycan epitope on a specific protein of interest (Aim 2b). The first class of imaging agents will be used to visualize levels of sialic acid, an important glycan associated with cancer progression and metastasis. The second class will be used to monitor sialic acid levels on v3 integrin. The sialylation of v3 integrin may play a vital role in promoting angiogenesis and metastasis, but is currently poorly understood. We will validate the new imaging instrumentation and imaging agents in orthotopic prostate cancer mouse models and study the spatiotemporal patterns of sialic acid and sialylated v3 integrin during prostate cancer progression and metastasis (Aim 3).